Universal amplification of fragmented RNA

ABSTRACT

The invention relates to methods of using fragmented RNA, such as RNA obtained from archived fixed paraffin-embedded tissue material (FPET RNA) or other clinically biopsied tissue specimens for universal gene expression profiling.

This application claims priority under 35 U.S.C. § 119(e) to provisionalapplication Ser. No. 60/532,684 filed on Dec. 23, 2003, the entiredisclosure of which is hereby expressly incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to methods of preparing RNA for geneexpression profiling by a variety of methods. The methods of theinvention are particularly useful for universal amplification of RNA,including RNA in which one or more RNA species is fragmented and/orblocked at it 3′ terminus, such as is obtained from fixedparaffin-embedded tissue (FPET). The methods are also useful fordetecting RNA species which lack polyadenylation. In addition, methodsof enhanced RT-PCR for useful in gene expression profiling are provided.

DESCRIPTION OF THE RELATED ART

Gene expression profiling is increasingly important both in biologicalresearch and in clinical practice. Gene expression profiling has beenused to classify various cancer types (see, e.g. Golub et al., Science286:531-537 (1999); Bhattacharjae et al., Proc. Natl. Acad. Sci. USA98:13790-13795 (2001); Chen-Hsiang et al., Bioinformatics 17 (Suppl.1):S316-S322 (2001); Ramaswamy et al., Proc. Natl. Acad. Sci. USA98:15149-15154 (2001); Martin et al., Cancer Res. 60:2232-2238 (2000);West et al., Proc. Natl. Acad. Sci. USA 98:11462-11467 (2001); Sorlie etal., Proc. Natl. Acad. Sci. USA 98:10869-10874 (2001); Yan et al.,Cancer Res. 61:8375-8380 (2001)), and to predict clinical outcome ofcancer, such as breast cancer (Van't Veer et al., Nature 415:530-536(2002) and lung cancer (Beer et al., Nat. Med. 8:816-24 (2002)).

The most commonly used methods known in the art for the quantificationof mRNA expression in a sample include northern blotting and in situhybridization (Parker & Barnes, Methods in Molecular Biology 106:247-283(1999)); RNAse protection assays (Hod, Biotechniques 13:852-854 (1992));microarrays (Schena et al., Proc. Natl. Acad. Sci. USA 93(2):106-149(1996)), and reverse transcription polymerase chain reaction (RT-PCR)(Weis et al., Trends in Genetics 8:263-264 (1992)). Alternatively,antibodies may be employed that can recognize specific duplexes,including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes orDNA-protein duplexes. Of these, due to its sensitivity, reproducibility,and large dynamic range, real-time RT-PCR is becoming the method ofchoice for high-throughput, accurate expression profiling.

In many situations where gene expression profiling is potentiallyuseful, there is insufficient material for analysis without prioramplification of RNA. As RNA cannot serve as a template for PCR, thefirst step in gene expression profiling by RT-PCR is the reversetranscription of the RNA template into cDNA, followed by its exponentialamplification in a PCR reaction. The conversion of mRNA to cDNA istypically performed by oligo dT priming of the mRNA in the presence ofthe reverse transcriptase (RT) enzyme. This step, however, is noteffective if the source of mRNA is a fixed, paraffin-embedded tissuesample, which may have been archived as long as 10-20 years, and inwhich the RNA is badly degraded (Lewis et al., J. Pathol. 195:66-71(2001)).

Because FPET samples are the most widely available source of RNA forgene expression profiling in clinical oncology and because archived FPETsamples are an important source of RNA for gene expression profiling inresearch oncology, there is a critical need for methods that enable andimprove the efficacy of gene expression profiling using such tissuesamples.

SUMMARY OF THE INVENTION

The present invention provides a sample preparation method that enablesglobal amplification of even very small or very fragmented RNA samples.The method of the invention improve the sensitivity of RNA analysismethods, including RT-PCR and hybridization arrays. Furthermore, themethods of the invention permit the measurement of mRNA levels of allexpressed genes including fragmented and/or blocked mRNA species inarchived paraffin-embedded tissue samples. This method also permits themeasurement of non-polyadenylated mRNA such as histones and non-codingRNAs, such as microRNAs (miRNAs). The invention may additionally includean enhanced reverse transcription step and a modified PCR step thatincreases the sensitivity of RT-PCR used for gene expression profilingof fragmented RNA samples.

In one aspect, the invention concerns a method for preparing amultiplicity of RNA species, which may include fragmented and/or blockedRNA species, for gene expression analysis comprising the steps of:

-   (a) polyadenylating the fragmented RNA, and-   (b) converting the polyadenylated fragmented RNA obtained in    step (a) to cDNA.

Typically, the size of the RNA species within the fragmented RNA isbetween about 20 bases and about 2000 bases, more frequently betweenabout 50 and about 300 bases.

Polyadenylation can, for example, be performed with E. coli polyApolymerase.

Since at least some RNA species within the fragmented RNA may be blockedat their 3′ termini, the method of the invention may additionallyinclude a step of deblocking. Deblocking can be performed by usingconventional reagents, such as, for example, with a phosphatase enzyme,e.g. calf alkaline phosphatase (CIP), bacterial alkaline phosphatase,shrimp alkaline phosphatase, or variants thereof, or with apolynucleotide kinase (PNK), e.g. T4 polynucleotide kinase (T4 PNK), orvariants thereof.

In one embodiment, the polyadenylated fragmented RNA obtained in step(a) above is converted to cDNA by treatment with a reverse transcriptaseand oligo-dT primers, where the oligo-dT primers may optionally containan RNA polymerase promoter (e.g. T7 RNA polymerase promoter) sequence.

In another embodiment, before converting the polyadenylated fragmentedRNA obtained in step (a) above to cDNA, the polyadenylating agent, suchas CIP, or PNK, is removed.

In a further embodiment, the polyadenylated fragmented RNA is convertedto cDNA without prior removal of the polyadenylating agent, such as CIP,or PNK.

In a still further embodiment, the polyadenylated fragmented RNA isenriched, e.g. by removal of rRNA sequences, prior to conversion to cDNAand subsequently to double-stranded cDNA.

In yet another embodiment, the polyadenylated fragmented RNA isimmobilized before conversion to a cDNA.

In a particular embodiment, the polyadenylated fragmented RNA ishybridized to a solid phase bead format. If desired, the immobilizedpolyadenylated fragmented RNA is enriched prior to conversion to cDNA.The enrichment may comprise removal of rRNA sequences by hybridizationto bead immobilized complementary rRNA oligonucleotides.

In yet another embodiment, the RNA is mRNA obtained from a fixed,paraffin-embedded tissue sample, such as a tumor sample, where the tumormay be cancer, such as, for example, breast cancer, colon cancer, lungcancer, prostate cancer, hepatocellular cancer, gastric cancer,pancreatic cancer, cervical cancer, ovarian cancer, liver cancer,bladder cancer, cancer of the urinary tract, thyroid cancer, renalcancer, carcinoma, melanoma, or brain cancer.

The method of the invention may additional comprise the step of (c) PCRamplification using one or more cDNA species present in the cDNAobtained in step (b) above as a template.

In a particular embodiment, PCR amplification comprises about 40 cycles,of which the first five cycles, or the first two to five cycles, or thefirst two cycles are performed at a lower annealing/extensiontemperature, such as, at a temperature of about 40° C. to 58° C., e.g.about 50° C.

In another embodiment, the method of the invention further comprises thesteps of:

-   (d) converting the cDNA obtained in step (b) to double-stranded DNA;    and-   (e) amplifying the RNA by subjecting the double-stranded DNA    obtained in step (d) to in vitro transcription with an RNA    polymerase to obtain amplified complementary RNA (cRNA).

In another embodiment of the method of the invention, the polyadenylatedfragmented RNA obtained in step (a) is converted to cDNA by treatmentwith a reverse transcriptase and extended reverse primers, and the cDNAobtained is amplified by PCR using a forward and a reverse PCR primerand a probe, designed based on a target amplicon.

In another aspect, the invention concerns a method for enhanced cDNAsynthesis, comprising converting RNA to cDNA by treatment with a reversetranscriptase and extended primers, and amplifying the cDNA obtained byPCR using a forward and a reverse PCR primer and a probe, designed basedon a target amplicon. The RNA may be fragmented, at least part of whichmay be non-polyadenylated.

In yet another aspect, the invention concerns a method for preparing RNAcomprising a multiplicity of RNA species for gene expression analysiscomprising the steps of:

-   (a) polyadenylating said RNA; and-   (b) converting the polyadenylated RNA to cDNA by reverse    transcriptase and oligo dT or oligo dT-T7 primers.

In a still further aspect, the invention concerns a method for preparingRNA comprising a multiplicity of RNA species for gene expressionanalysis comprising the steps of:

-   (a) polyadenylating said RNA; and-   (b) converting the polyadenylated RNA to cDNA by reverse    transcriptase and oligo dT-T7 primers containing a T7 RNA polymerase    promoter, and-   (c) subjecting the double-stranded DNA obtained in step (b) to in    vitro transcription with a T7 RNA polymerase to obtain amplified    complementary RNA (cRNA).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chart illustrating the overall workflow of the amplificationprocess in the invention used for measuring gene expression. In thisFigure, FPET stands for “fixed paraffin-embedded tissue, PNK stands for“polynucleotide kinase”, CIP stands for “calf intestinal alkalinephosphatase”, EPAP stands for “E. coli polyA polymerase”, TdT stands for“Terminal Transferase”, IVT stands for “in vitro transcription”, rNTPstands for “ribonucleoside-5′-triphosphate”, dNTP stands for“2′-deoxyribonucleoside-5′-triphosphate”, ATP stands for“adenosine-5′-triphosphate”. T7-(T)₂₄ is a primer for cDNA synthesisusing reverse transcriptase and stands for T7 RNA polymerase promotersequence attached to the 5′end of polydeoxyadenylate. T7-(N)15 is aprimer for cDNA synthesis using reverse transcriptase and stands for T7RNA polymerase promoter sequence attached to the 5′ end of a randomdeoxy-pentadecamer. In FIG. 1, there are three representative protocols(A, B and C) of the invention. Processes A and B start with FPET RNA andinvolve 1) direct or indirect unblocking of the 3′OH on the terminalnucleotide, 2) poly A tailing of the 3′ end, 3) oligo dT-primeddouble-strand cDNA synthesis with the incorporation of a T7 RNApolymerase promoter, and 4) RNA amplification by in vitro transcription.Process C (middle of diagram) starts with FPET RNA and involves 1)directly synthesizing double-stranded cDNA using T7-(N)15 primers with aT7 RNA polymerase promoter, and 2) RNA amplification by in vitrotranscription.

FIG. 2 shows a gel image of FPET RNA isolated from twelve differentbreast cancer patient's biopsies. Lanes M1 and M2 show RNA markers withthe size of each band denoted in bases. Lanes 1-4, 5-8 and 9-12 are fromtumor biopsies that have been archived for 1, 6 and 17 yearsrespectively. Samples were analyzed by capillary electrophoresis on anAgilent 2100 Bioanalyzer using an RNA 6000 Nanochip.

FIG. 3 shows selected gene expression analysis of breast tumor FPET RNA,randomly degraded breast tumor RNA and intact breast tumor RNA.Expression was analyzed by real time quantitative RT-PCR (TaqMan®) on anABI Prism® 7700. cDNA synthesis was primed with either gene specificprimers (GSP) or oligo (dT)₁₂₋₁₈. Relative yields are measured by thethreshold cycle (Ct).

FIG. 4 shows a selected gene expression analysis of breast tumor FPETRNA. Expression was analyzed by real time quantitative RT-PCR (TaqMan®)on an ABI Prism® 7700. cDNA synthesis was primed with either GSPs oroligo (dT)₁₂₋₁₈ using template RNA that was not polyadenylated (FPET/GSPand FPET/dT) or cDNA synthesis was primed with oligo (dT)₁₂₋₁₈ usingtemplate RNA that was polyadenylated (FPET-pA/dT). Relative yields aremeasured by the threshold cycle (Ct).

FIG. 5 shows a gene expression analysis of in vitro transcribed cRNAs.Expression was analyzed by real time quantitative RT-PCR (TaqMan®) on anABI Prism® 7700. RNA polyadenylation was performed with 0.2 units ofEPAP and cDNA synthesis was performed as in FIG. 4. See Materials andMethods for generating the template cRNA. Relative yields are measuredby the threshold cycle (Ct). Inset: Agilent 2100 gel image of cRNAstreated with 0, 0.1 or 0.2 units of EPAP. Lane M shows RNA markers withthe size of each band denoted in bases.

FIG. 6A shows a gel image of FPET RNAs that have been treated with PNK(lane 1), PNK buffer control (lane 2), CIP (lane 3) or CIP buffercontrol (lane 4). FIG. 6B shows a gel image of FPET RNAs that have beentreated with PNK buffer control followed by EPAP buffer control (lane1), PNK followed by EPAP buffer control (lane 2), PNK buffer controlfollowed by EPAP, or PNK followed by EPAP (lane 4). Samples wereanalyzed by capillary electrophoresis on an Agilent 2100 using an RNA6000 Nanochip. Lanes M1 and M2 show RNA markers with the size of eachband denoted in bases.

FIG. 7 shows a selected gene expression analysis of breast tumor FPETRNA. Expression was analyzed by real time quantitative RT-PCR (TaqMan®)on an ABI Prism® 7700. Prior to gene expression analysis, the RNA wastreated with PNK (+PNK) or buffer control (−PNK), followed bypolyadenylation by EPAP. The RNA was then converted into cDNA withreverse transcriptase and oligo dT primers (+PNK/Oligo dT and −PNK/OligodT) or gene specific primers (+PNK/GSP and −PNK/GSP). Relative yieldsare measured by the threshold cycle (Ct). Inset: Agilent 2100 gel imageof FPET RNA treated with PNK or buffer control followed by EPAP. Lane Mshows RNA markers with the size of each band denoted in bases.

FIG. 8 shows selected gene expression analysis of breast tumor FPET RNA.Expression was analyzed by real time quantitative RT-PCR (TaqMan®) on anABI Prism® 7700. Prior to gene expression analysis, the RNA was treatedwith CIP (+CIP) or buffer control (−CIP), followed by polyadenylation byEPAP. The RNA was then converted into cDNA with reverse transcriptaseand oligo dT primers (+CIP/Oligo dT and −CIP/Oligo dT) or gene specificprimers (+CIP/GSP and −CIP/GSP). Relative yields are measured by thethreshold cycle (Ct). Inset: Agilent 2100 gel image of FPET RNA treatedwith CIP or buffer control followed by EPAP. Lane M shows RNA markerswith the size of each band denoted in bases.

FIG. 9 shows a 96 gene panel expression analysis of breast tumor FPETRNA. Expression was analyzed by real time quantitative RT-PCR (TaqMan®)on an ABI Prism® 7900. Prior to gene expression analysis, the RNA wastreated with PNK, followed by treatment with EPAP. The RNAs were thenconverted into cDNA with reverse transcriptase and oligo dT primers(pA_dT) or gene specific primers (pA_GSP1). Non-polyadenylated RNA wasalso converted to cDNA as above (untreated_dT and untreated GSP1).Relative yields are measured by the threshold cycle (Ct). Statisticalanalysis of the results are shown in Table 1.

FIGS. 10A-C show a 46 gene panel expression analysis of amplified andunamplified FPET RNA. Three different RNAs were profiled. IntactUniversal total RNA (Stratagene, La Jolla, Calif.) is shown in FIG. 10A,placental FPET RNA (from placenta treated for 1.5-2 h with formalin andparaffin-embedded by BioPathology Sciences Medical Corporation, SouthSan Francisco, Calif.) is shown in FIG. 10B and breast tumor FPET RNA(Clinomics BioSciences, Pittsfield, Mass.) is shown in FIG. 10C.Expression was analyzed by real time quantitative RT-PCR (TaqMan®) on anABI Prism® 7900. Prior to gene expression analysis, two separate samplesof RNA were treated with PNK followed by EPAP and then converted intocDNA with reverse transcriptase and oligo dT-T7 primers. The cDNA wasmade double-stranded with DNA polymerase I and RNAseH, amplified by IVT[IVT (tailed-1) and IVT (tailed-2)] and then analyzed by TaqMan® As acontrol, non PNK/nonEPAP-treated FPET RNA was converted todouble-stranded cDNA and amplified by IVT [IVT (untailed)] as above orconverted to cDNA by GSPs (GSP) prior to analysis by TaqMan®. Relativeyields are measured by the threshold cycle (Ct). The inset tables showthe overall average Ct of the 46 genes profiled for each RNA sample.Also shown are the Pearson correlation coefficients (R) between theunamplified (GSP) and amplified (IVT) RNA samples for the 46 profiledgenes.

FIG. 11 is a scheme depicting the strategy for enhancing gene specificpriming of fragmented FPET RNA. In this Figure, FPET stands for “fixedparaffin-embedded tissue, ^(Me) Gppp refers to the 5′ methylatedguanylate CAP structure of mRNA, A₁₀₀₋₃₀₀ refers to the 3′ polyA tractof mRNA, RT stands for reverse transcriptase, PCR stands for polymerasechain reaction, amplicon stands for the region of the mRNA amplified byPCR and GSP stands for gene specific primer. The extended reverseprimers (10b, 20b and 30b) are identical to the reverse primer butextend roughly 10, 20 and 30 bases further into the amplicon. The 3′ ofthe 30b RT primer and the 3′ end of the forward primer are separated bya single base.

FIG. 12 shows selected gene expression analysis of breast tumor FPET RNA(Clinomics 168). Expression was analyzed by real time quantitativeRT-PCR (TaqMan®) on an ABI Prism® 7700. cDNA synthesis was primed witheither gene specific primers (Std primers) or extended reverse primers(10b, 20b and 30b primers). Relative yields are measured by thethreshold cycle (Ct).

FIG. 13 is a schematic diagram illustrating the overall workflow of theimproved, bead based amplification process in the invention used formeasuring gene expression. In this figure, FPET stands for “fixedparaffin-embedded tissue, PNK stands for “polynucleotide kinase”, EPAPstands for “E. coli polyA polymerase”, O-T7-(TTT) is a solid phase,immobilized primer for cDNA synthesis consisting of a T7 RNA polymerasepromoter sequence and an oligodeoxythymidylate sequence attached to amagnetic polystyrene bead, RT stands for “Reverse Transcriptase”, RNAseH stands for “Ribonuclease H”, DNA pol I stands for the “DNA polymeraseI”, IVT stands for “in vitro transcription”, cRNA stands for“complementary RNA generated by IVT, and O-(−) rRNA refers to beadimmobilized complementary ribosomal RNA sequences (synthesized as shortDNA oligos). The improved, bead based protocol is shown in the centralportion of the diagram (solid arrows). The process starts with FPET RNA,generally 50-200 ng, and involves 1) unblocking of the 3′OH on theterminal nucleotide with PNK, 2) direct EPAP poly A tailing of the FPETRNA 3′ without cleanup from PNK step, 3) hybridization of polyadenylatedFPET RNA to oligo dT-T7 RNA polymerase promoter sequences immobilized tobeads followed by 4) cDNA synthesis with RT, 5) partial RNA degradationby RNAse H and second strand DNA synthesis with DNA polymerase I and 6)RNA amplification by in vitro transcription. An optional procedure for asecond round of IVT is shown in step 7 (broken arrows). Another optionalstep, shown in step 2′ involves depletion of ribosomal rRNA fragments(dotted arrow).

FIG. 14 shows the results from various PNK cleanup modificationsfollowed by polyadenylation by EPAP. The left panel shows FPET RNA sizeby microcapillary electrophoresis using the Agilent 2100 Bioanalyzer.Lane descriptions: Ladder, RNA molecular weight markers from 200 bases(lowest) to 6000 bases; SM, unmodified FPET RNA. 1-6, PNK cleanupconditions described in the right panel. P/C refers to phenol/chloroform; DEPC-30 column refers to CHROMA SPIN™ DEPC-H₂O 30 column, heatinactivation conditions were 65° C., 20 min. Percent recovery after EPAPcleanup is relative to input FPET RNA (1000 ng).

FIG. 15 shows a 47 gene panel expression analysis of amplified breasttumor FPET RNA (Clinomics BioSciences, Pittsfield, Mass.). Expressionwas analyzed by real time quantitative RT-PCR (TaqMan®) on an ABI Prism®7900. Relative yields are measured by the threshold cycle (Ct). Theinset tables show the overall average Ct of the 47 genes profiled foreach cleanup condition and the cRNA yield for each IVT. The cleanupconditions are described below in Example 2.

FIG. 16 shows a 47 gene panel expression analysis of Placental FPET RNAamplified by a non-bead based (Free IVT) and a bead based (Solid PhaseIVT) described in Example 3. For comparison, the profile of unamplifiedstarting material (SM) is also shown. The placenta was treated for 1.5-2h with formalin and paraffin-embedded by BioPathology Sciences MedicalCorporation, South San Francisco, Calif. Expression was analyzed by realtime quantitative RT-PCR (TaqMan®) on an ABI Prism® 7900. Relativeyields are measured by the threshold cycle (Ct). The inset tables showthe overall average Ct of the 47 genes profiled for each cleanupcondition and the cRNA yield for each IVT. Also shown are yields afternormalization (Yield;Ct. adj.). The equation for normalizing yields is:cRNA Yield/2^((Avg. Ct.-SM Ct.))

FIGS. 17A-C show a 47 gene panel expression analysis of amplifiedPlacental FPET RNA. The placenta was treated for 1.5-2 h with formalinand paraffin-embedded by BioPathology Sciences Medical Corporation,South San Francisco, Calif. Expression was analyzed by real timequantitative RT-PCR (TaqMan®) on an ABI Prism® 7900. Relative yields aremeasured by the threshold cycle (Ct). The inset tables show the overallaverage Ct of the 47 genes profiled for each FRA condition and the cRNAyield for each IVT. The three FRA conditions are described below inExample 4. FIGS. 17A and 17B show the results from the primary andsecondary amplification, respectively. FIG. 17C shows a comparison of aprimary and secondary IVT (Condition 3).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

A. Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton et al., Dictionary ofMicrobiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York,N.Y. 1994), provide one skilled in the art with a general guide to manyof the terms used in the present application.

The term “polyadenylation” or “poly A tailing” refers to the addition ofa stretch of adenylate molecules (poly (A)) to the 3′ end of RNA, e.g.mRNA.

“Efficiency of polyadenylation” refers to the ease with which poly Aaddition occurs and is dependent upon the availability of the free3′-hydroxyl (3′ OH) group at the 3′-terminal ribose moiety of RNA.

“Blocking of polyadenylation” or “blocked 3′ end” of RNA refers toblocking the availability of the 3′-terminal ribose moiety of RNA for apolyadenylation reaction. This may occur, for example, because the 3′terminus of RNA contains various phosphate esters typically 2′-3′ cyclicphosphates, 2′-monophosphates and 3′-monophosphates which need to beremoved to permit addition of a poly A tail to the 3′ terminus of theRNA.

“Dephosphorylation” refers to the removal of phosphate esters (describedabove) by any methods, including but not limited to enzymatictechniques, such as using calf intestinal phosphatase (CIP) or T4Polynucleotide Kinase (PNK).

“Threshold cycle (Ct)” refers to relative yields of nucleic acidamplified in a PCR reaction. During TaqMan PCR, the 5′-nuclease activityof the Taq polymerase is utilized to cleave and release a quenchedreporter dye present on a third oligonucleotide primer (which isnon-extendible by Taq polymerase) that detects a nucleotide sequencelocated between the two PCR primers. One molecule of reporter dye isliberated for each newly synthesized nucleic acid molecule and detectionof this unquenched reporter dye provides the basis for quantitativeinterpretation of amplification or the mount of product amplified tothat point in the amplification reaction. The point when the fluorescentsignal is first recorded as statistically significant is the thresholdcycle (Ct). The lower the Ct value, the more abundant the mRNA is andthe better the performance of the nucleic acid (cDNA or poly A mRNA ordegraded mRNA) in the expression profiling array.

The term “microarray” refers to an ordered arrangement of hybridizablearray elements, such as polynucleotide probes, on a substrate.

The term “polynucleotide,” when used in singular or plural, generallyrefers to any polyribonucleotide or polydeoxribonucleotide, which may beunmodified RNA or DNA or modified RNA or DNA. Thus, for instance,polynucleotides as defined herein include, without limitation, single-and double-stranded DNA, DNA including single- and double-strandedregions, single- and double-stranded RNA, and RNA including single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded or includesingle- and double-stranded regions. In addition, the term“polynucleotide” as used herein refers to triple-stranded regionscomprising RNA or DNA or both RNA and DNA. The strands in such regionsmay be from the same molecule or from different molecules. The regionsmay include all of one or more of the molecules, but more typicallyinvolve only a region of some of the molecules. One of the molecules ofa triple-helical region often is an oligonucleotide. The term“polynucleotide” specifically includes DNAs and RNAs that contain one ormore modified bases. Thus, DNAs or RNAs with backbones modified forstability or for other reasons are “polynucleotides” as that term isintended herein. Moreover, DNAs or RNAs comprising unusual bases, suchas inosine, or modified bases, such as tritiated bases, are includedwithin the term “polynucleotides” as defined herein. In general, theterm “polynucleotide” embraces all chemically, enzymatically and/ormetabolically modified forms of unmodified polynucleotides, as well asthe chemical forms of DNA and RNA characteristic of viruses and cells,including simple and complex cells.

The term “oligonucleotide” refers to a relatively short polynucleotide,including, without limitation, single-stranded deoxyribonucleotides,single- or double-stranded ribonucleotides, RNA:DNA hybrids anddouble-stranded DNAs. Oligonucleotides, such as single-stranded DNAprobe oligonucleotides, are often synthesized by chemical methods, forexample using automated oligonucleotide synthesizers that arecommercially available. However, oligonucleotides can be made by avariety of other methods, including in vitro recombinant DNA-mediatedtechniques and by expression of DNAs in cells and organisms.

The terms “differentially expressed gene,” “differential geneexpression” and their synonyms, which are used interchangeably, refer toa gene whose expression is activated to a higher or lower level in asubject suffering from a disease, specifically cancer, such as breastcancer, relative to its expression in a normal or control subject. Theterms also include genes whose expression is activated to a higher orlower level at different stages of the same disease. It is alsounderstood that a differentially expressed gene may be either activatedor inhibited at the nucleic acid level or protein level, or may besubject to alternative splicing to result in a different polypeptideproduct. Such differences may be evidenced by a change in mRNA levels,surface expression, secretion or other partitioning of a polypeptide,for example. Differential gene expression may include a comparison ofexpression between two or more genes, or a comparison of the ratios ofthe expression between two or more genes, or even a comparison of twodifferently processed products of the same gene, which differ betweennormal subjects and subjects suffering from a disease, specificallycancer, or between various stages of the same disease. Differentialexpression includes both quantitative, as well as qualitative,differences in the temporal or cellular expression pattern in a gene orits expression products among, for example, normal and diseased cells,or among cells which have undergone different disease events or diseasestages. For the purpose of this invention, “differential geneexpression” is considered to be present when there is at least abouttwo-fold, preferably at least about four-fold, more preferably at leastabout six-fold, most preferably at least about ten-fold differencebetween the expression of a given gene in normal and diseased subjects,or in various stages of disease development in a diseased subject.

The phrase “gene amplification” refers to a process by which multiplecopies of a gene or gene fragment are formed in a particular cell orcell line. The duplicated region (a stretch of amplified DNA) is oftenreferred to as “amplicon.” Usually, the amount of the messenger RNA(mRNA) produced, i.e., the level of gene expression, also increases inthe proportion of the number of copies made of the particular geneexpressed.

The terms “splicing” and “RNA splicing” are used interchangeably andrefer to RNA processing that removes introns and joins exons to producemature mRNA with continuous coding sequence that moves into thecytoplasm of an eukaryotic cell.

In theory, the term “exon” refers to any segment of an interrupted genethat is represented in the mature RNA product (B. Lewin. Genes IV CellPress, Cambridge Mass. 1990). In theory the term “intron” refers to anysegment of DNA that is transcribed but removed from within thetranscript by splicing together the exons on either side of it.Operationally, exon sequences occur in the mRNA sequence of a gene asdefined by Ref. Seq ID numbers. Operationally, intron sequences are theintervening sequences within the genomic DNA of a gene, bracketed byexon sequences and having GT and AG splice consensus sequences at their5′ and 3′ boundaries.

The term “tumor,” as used herein, refers to all neoplastic cell growthand proliferation, whether malignant or benign, and all pre-cancerousand cancerous cells and tissues.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, breast cancer, colon cancer, lung cancer, prostate cancer,hepatocellular cancer, gastric cancer, pancreatic cancer, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, cancer of theurinary tract, thyroid cancer, renal cancer, carcinoma, melanoma, andbrain cancer.

The “pathology” of cancer includes all phenomena that compromise thewell-being of the patient. This includes, without limitation, abnormalor uncontrollable cell growth, metastasis, interference with the normalfunctioning of neighboring cells, release of cytokines or othersecretory products at abnormal levels, suppression or aggravation ofinflammatory or immunological response, etc.

B. Detailed Description

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, and biochemistry,which are within the skill of the art. Such techniques are explainedfully in the literature, such as, “Molecular Cloning: A LaboratoryManual”, 2 ^(nd) edition (Sambrook et al., 1989); “OligonucleotideSynthesis” (M. J. Gait, ed., 1984); “Animal Cell Culture” (R. I.Freshney, ed., 1987); “Methods in Enzymology” (Academic Press, Inc.);“Handbook of Experimental Immunology”, 4 ^(th) edition (D. M. Weir & C.C. Blackwell, eds., Blackwell Science Inc., 1987); “Gene TransferVectors for Mammalian Cells” (J. M. Miller & M. P. Calos, eds., 1987);“Current Protocols in Molecular Biology” (F. M. Ausubel et al., eds.,1987); and “PCR: The Polymerase Chain Reaction”, (Mullis et al., eds.,1994). Specific protocols are described in the Materials and Methodsection of the Example below.

As discussed earlier, gene expression profiling has become an importanttool of biological research and clinical practice.

Real-Time Reverse Transcriptase PCR (RT-PCR)

Of the gene expression profiling techniques, the most sensitive and mostflexible quantitative method is RT-PCR, which can be used to comparemRNA levels in different sample populations, in normal and tumortissues, with or without drug treatment, to characterize patterns ofgene expression, to discriminate between closely related mRNAs, and toanalyze RNA structure.

The first step in gene expression profiling by RT-PCR is the reversetranscription of the RNA template into cDNA, followed by its exponentialamplification in a PCR reaction. The two most commonly used reversetranscriptases are avian myeloblastosis virus reverse transcriptase(AMV-RT) and Moloney murine leukemia virus reverse transcriptase(MMLV-RT). The reverse transcription step is typically primed using genespecific primers, random hexamers, or oligo-dT primers, depending on thecircumstances and the goal of expression profiling. For example,extracted RNA can be reverse-transcribed using a GeneAmp RNA PCR kit(Perkin Elmer, Calif., USA), following the manufacturer's instructions.The derived cDNA can then be used as a template in the subsequent PCRreaction.

Although the PCR step can use a variety of thermostable DNA-dependentDNA polymerases, it typically employs the Taq DNA polymerase, which hasa 5′-3′ nuclease activity but lacks a 3′-5′ proofreading endonucleaseactivity. Thus, TaqMan® PCR typically utilizes the 5′-nuclease activityof Taq or Th polymerase to hydrolyze a hybridization probe bound to itstarget amplicon, but any enzyme with equivalent 5′ nuclease activity canbe used. Two oligonucleotide primers are used to generate an amplicontypical of a PCR reaction. A third oligonucleotide, or probe, isdesigned to detect nucleotide sequence located between the two PCRprimers. The probe is non-extendible by Taq DNA polymerase enzyme, andis labeled with a reporter fluorescent dye and a quencher fluorescentdye. Any laser-induced emission from the reporter dye is quenched by thequenching dye when the two dyes are located close together as they areon the probe. During the amplification reaction, the Taq DNA polymeraseenzyme cleaves the probe in a template-dependent manner. The resultantprobe fragments disassociate in solution, and signal from the releasedreporter dye is free from the quenching effect of the secondfluorophore. One molecule of reporter dye is liberated for each newmolecule synthesized, and detection of the unquenched reporter dyeprovides the basis for quantitative interpretation of the data.

TaqMan® RT-PCR can be performed using commercially available equipment,such as, for example, ABI PRISM 7700™ Sequence Detection System™(Perkin-Elmer-Applied Biosystems, Foster City, Calif., USA), orLightcycler (Roche Molecular Biochemicals, Mannheim, Germany). In apreferred embodiment, the 5′ nuclease procedure is run on a real-timequantitative PCR device such as the ABI PRISM 7700™ Sequence DetectionSystem™. The system consists of a thermocycler, laser, charge-coupleddevice (CCD), camera and computer. The system amplifies samples in a96-well format on a thermocycler. During amplification, laser-inducedfluorescent signal is collected in real-time through fiber optics cablesfor all 96 wells, and detected at the CCD. The system includes softwarefor running the instrument and for analyzing the data.

5′-Nuclease assay data are initially expressed as Ct, or the thresholdcycle. As discussed above, fluorescence values are recorded during everycycle and represent the amount of product amplified to that point in theamplification reaction. The point when the fluorescent signal is firstrecorded as statistically significant is the threshold cycle (C_(t)).

To minimize errors and the effect of sample-to-sample variation, RT-PCRis usually performed using an internal standard. The ideal internalstandard is expressed at a constant level among different tissues, andis unaffected by the experimental treatment. RNAs most frequently usedto normalize patterns of gene expression are mRNAs for the housekeepinggenes glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and β-actin.

A more recent variation of the RT-PCR technique is the real-timequantitative PCR, which measures PCR product accumulation through adual-labeled fluorigenic probe (i.e., TaqMan® probe). Real time PCR iscompatible both with quantitative competitive PCR, where internalcompetitor for each target sequence is used for normalization, and withquantitative comparative PCR using a normalization gene contained withinthe sample, or a housekeeping gene for RT-PCR. For further details see,e.g. Held et al., Genome Research 6:986-994 (1996).

Microarray Analysis

Often another method of choice for gene expression profiling is themicroarray technique. In this method, polynucleotide sequences ofinterest (including cDNAs and oligonucleotides) are plated, or arrayed,on a microchip substrate. The arrayed sequences are then hybridized withspecific DNA probes from cells or tissues of interest. Just as in theRT-PCR method, the source of mRNA typically is total RNA isolated fromhuman tumors or tumor cell lines, and corresponding normal tissues orcell lines. Thus RNA can be isolated from a variety of primary tumors ortumor cell lines. If the source of mRNA is a primary tumor, mRNA can beextracted, for example, from frozen or archived paraffin-embedded andfixed (e.g. formalin-fixed) tissue samples, which are routinely preparedand preserved in everyday clinical practice.

In a specific embodiment of the microarray technique, PCR amplifiedinserts of cDNA clones are applied to a substrate in a dense array.Preferably at least 10,000 nucleotide sequences are applied to thesubstrate. The microarrayed genes, immobilized on the microchip at10,000 elements each, are suitable for hybridization under stringentconditions. Fluorescently labeled cDNA probes may be generated throughincorporation of fluorescent nucleotides by reverse transcription of RNAextracted from tissues of interest. Labeled cDNA probes applied to thechip hybridize with specificity to each spot of DNA on the array. Afterstringent washing to remove non-specifically bound probes, the chip isscanned by confocal laser microscopy or by another detection method,such as a CCD camera. Quantitation of hybridization of each arrayedelement allows for assessment of corresponding mRNA abundance. With dualcolor fluorescence, separately labeled cDNA probes generated from twosources of RNA are hybridized pairwise to the array. The relativeabundance of the transcripts from the two sources corresponding to eachspecified gene is thus determined simultaneously. The miniaturized scaleof the hybridization affords a convenient and rapid evaluation of theexpression pattern for large numbers of genes. Such methods have beenshown to have the sensitivity required to detect rare transcripts, whichare expressed at a few copies per cell, and to reproducibly detect atleast approximately two-fold differences in the expression levels(Schena et al., Proc. Natl. Acad. Sci. USA 93(2):106-149 (1996)).Microarray analysis can be performed by commercially availableequipment, following manufacturer's protocols, such as by using theAffymetrix GenChip technology, or Incyte's microarray technology.

The development of microarray methods for large-scale analysis of geneexpression makes it possible to search systematically for molecularmarkers of cancer classification and outcome prediction in a variety oftumor types.

RNA Extraction and Amplification for Gene Expression Profiling

A common step in gene expression profiling by the RT-PCR and microarraytechniques is the extraction of mRNA from biological samples.

General methods for mRNA extraction are well known in the art and aredisclosed in standard textbooks of molecular biology, including Ausubelet al., Current Protocols of Molecular Biology, John Wiley and Sons(1997). Methods for RNA extraction from paraffin embedded tissues aredisclosed, for example, in Rupp and Locker, Lab Invest. 56:A67 (1987),and De Andrés et al., BioTechniques 18:42044 (1995). In particular, RNAisolation can be performed using purification kit, buffer set andprotease from commercial manufacturers, such as Qiagen, according to themanufacturer's instructions. For example, total RNA from cells inculture can be isolated using Qiagen RNeasy mini-columns. Othercommercially available RNA isolation kits include MasterPure™ CompleteDNA and RNA Purification Kit (EPICENTRE®, Madison, Wis.), and ParaffinBlock RNA Isolation Kit (Ambion, Inc.). Total RNA from tissue samplescan be isolated using RNA Stat-60 (Tel-Test). RNA prepared from tumorcan be isolated, for example, by cesium chloride density gradientcentrifugation.

If necessary, DNA can be removed at various stages of RNA isolation, byDNase or other techniques well known in the art. After analysis of theRNA concentration after purification, RNA repair and/or amplificationsteps may be necessary before subjecting the RNA to any known expressiongene profiling methods, including RT-PCR coupled with 5′ exonuclease ofreporter probes (TaqMan® type assays), flap endonuclease assays(Cleavase and Invader type assays), oligonucleotide hybridizationarrays, cDNA hybridization arrays, oligonucleotide ligation assays, 3′single nucleotide extension assays and other assays designed to assessthe abundance of specific mRNA sequences in a biological sample.

Despite the availability of commercial products, and the extensiveknowledge available concerning the isolation of RNA from tissues,isolation of nucleic acid (RNA) from fixed, paraffin-embedded tissuespecimens (FPET) and its use for gene expression profiling is notwithout difficulty.

mRNA is notoriously difficult to extract and maintain in its nativestate, consequently, mRNA recovered from various biological sources, andspecifically from archived, fixed paraffin-embedded tissue (FPET) isoften fragmented and/or degraded. FIG. 2 shows an example of RNAisolated from formalin-fixed, paraffin embedded (PFE) breast cancersamples that were archived from 1 to 17 years. RNA degradationprogresses with archive storage time and results in RNA having anaverage size of about 100 bases after 17 years of storage. Bycomparison, intact mRNA has an average size of about 1800-2000 bases.

As discussed above, the extraction of mRNA is typically followed byconversion to cDNA using the primer dependent enzyme reversetranscriptase (RT). Universal conversion of intact mRNA to cDNA isperformed efficiently by oligo dT priming of the mRNA in the presence ofRT.

Effective priming with oligo dT during the PCR reaction is made possibleby the presence of a poly A tract at the 3′ end of mRNA. FIG. 3 showsthat intact mRNA can be efficiently profiled by TaqMan analysis usingcDNA generated by RT and oligo dT priming. As can also be seen,profiling of fixed, paraffin-embedded (FPET) or randomly degraded RNA(obtained by alkaline hydrolysis) by oligo dT primed cDNA synthesis isextremely inefficient as judged by the lower TaqMan signal (higher Ct)obtained relative to intact RNA. For the genes profiled, the signalsfrom the intact RNA are on the average 500-1000-times greater than thecorresponding signals from FPET RNA.

The present inventors have considered that inefficient conversion ofFPET mRNA to cDNA by oligo dT priming might result from the fact thatthe majority of the degraded mRNAs does not contain a polyA tail.Importantly, FIG. 3 also shows that degraded RNA can be efficientlyprofiled using gene-specific primers (GSP). This indicates that mostregions of the expressed genes are present in the randomly fragmentedRNA in proportions expected for the intact mRNA. This result suggeststhat it should be possible to perform effective universal geneexpression profiling on fragmented, e.g. FPET mRNA extracts.

To this end, it has been attempted to globally reverse transcribe FPETRNA by first polyadenylating the RNA and then performing oligo dT primedRT. As shown in FIG. 4, polyadenylation of FPET RNA prior to oligo dTpriming increased the conversion of the RNA to cDNA by about 2-4-fold asjudged by TaqMan analysis of 3 mRNAs. This result has been interpretedto suggest that polyadenylation may be a useful method to preparefragmented, e.g. FPET RNA for global reverse transcription andsubsequent gene expression profiling. However, this signal amplificationwas still only a small fraction of that obtained by priming with genespecific primers (GSP), which is the most efficient currently usedmethod of priming the conversion of selected regions of mRNA to cDNA byRT.

One recognition underlying the present invention is that the limitedconversion of fragmented RNA to cDNA is due to the fact that infragmented RNA, the 3′ end of a large proportion of RNA fragments isblocked and therefore not accessible to polyadenylation.

A model system has been developed to monitor the level ofpolyadenylation of fragmented, e.g. FPET RNA and determine its effect ongene expression profiling when RNA 3′ ends are not blocked. RNAfragments (˜100 bases) of three genes were generated by in vitrotranscription (IVT), then pooled and polyadenylated. Polyadenylation wasmonitored by capillary electrophoresis on the Agilent 2100 BioAnalyzer.The FIG. 5 inset demonstrates that tailing of RNA with 0.1 and 0.2 unitsof E. coli polyA polymerase (EPAP) adds between 20 and 200 adenylates tothe RNA. The polyadenylated RNA was then reverse transcribed to cDNAusing oligo dT priming and assayed by TaqMan analysis (FIG. 5). As canbe seen, polyadenylation of the RNA (0.2 EPAP/oligo-dT) resulted in adramatic increase in TaqMan signal relative to non-tailed RNA.

Based on the above result, it has been hypothesized that most 3′ ends inthe fragmented FPET RNA were blocked, probably due to enzymatichydrolysis with cellular RNAses that commonly yield 3′ PO₄ or cyclic2′-3′ PO₄. These modifications would effectively block thepolyadenylation of FPET RNA.

According to a variation of the method of the invention, effectiveconversion of fragmented mRNA to cDNA starts with the unblocking of the3′ terminus of the RNA. Any phosphatase, like calf alkaline phosphatase(CIP) or T4 polynucleotide kinase (PNK) can be used to remove 2′-3′cyclic phosphates, 2′-monophosphates and 3′-monophosphates, generatedtypically on the 3′ terminal ribose moiety of degraded RNAs. Thisensures efficient poly A addition by poly A polymerase to the 3′terminus of the FPET RNA. PNK, which is also a 3′ phosphatase, catalyzesthe hydrolysis of 3′-phosphoryl groups of deoxynucleoside3′-monophosphates, deoxynucleoside 3′-, 5′diphosphates and of3′-phosphoryl-polynucleotides. Other phosphatases like bacterialalkaline phosphatase, shrimp alkaline phosphatase, and derivativesthereof, can also be used to carry out such dephosphorylation reactions.The 3′ terminus now has a free 3′OH available.

Polyadenylation or “poly A tailing” of mRNA or FPET mRNA after enzymaticreactions like dephosphorylation involves appending of adenylatemolecules or poly (A) to the 3′ OH end of the RNA. In one embodiment,this is done using E. coli poly A polymerase. However, as will beunderstood by those skilled in the art, other poly A polymerases canalso be used.

Specific Embodiments

Three representative protocols (A, B and C) of the invention areillustrated in FIG. 1. Processes A and B start with FPET RNA andinvolve 1) direct or indirect unblocking of the 3′ OH on the terminalnucleotide, 2) poly A tailing of the 3′end, 3) poly dT-primeddouble-strand cDNA synthesis with the incorporation of a T7 RNApolymerase promoter, and 4) RNA amplification by in vitro transcription.

Process C (central arrow in the diagram) starts with FPET RNA andinvolves 1) T7-(N)15 primed double-stranded cDNA synthesis with theincorporation of a T7 RNA polymerase promoter, and 2) RNA amplificationby in vitro transcription.

Specifically, Protocol A involves a random primed (hexamers) cDNAsynthesis that generates cDNA with a free 3′ OH on the terminalnucleotide of the FPET cDNA. The FPET cDNA is then tailed with TerminalTransferase (TdT) and dATP. The poly dA-tailed cDNA is then converted todouble-stranded DNA with DNA polymerase I (Klenow) and T7-(dT)₂₄ primer.This material is then amplified with T7 RNA polymerase and rNTPs togenerate FPET RNA (+strand). This material is suitable for geneexpression analysis by TaqMan®. In order to be a suitable target formicroarrays containing (+) strand probes, the FPET RNA needs to beconverted to (−) strand cDNA in the presence of biotinylated dNTPs,random primers and RT. This material is now suitable for hybridizationto microarrays in order to perform universal gene expression profiling.For microarrays containing double-stranded probes, this final step isnot necessary. In this case, the IVT step should include biotin-rNTP'sas in protocol B (below).

Protocol B involves an unblocking of the 2′ OH and/or 3′OH of the3′-terminal nucleotide of FPET RNA with polynucleotide kinase (PNK) or[pH2 treatment (0.01M HCl or maleic acid)] followed by calf intestinalalkaline phosphatase (CIP). This enables the RNA to be efficientlypolyadenylated at the 3′ terminal nucleotide with E. coli polyApolymerase and ATP. Following polyadenylation, the RNA is converted tocDNA by reverse transcriptase using either oligo dT or oligo dT-T7 asprimers. The oligo dT primed cDNA can be directly used for geneexpression analysis by TaqMan® analysis. This method is preferred if theamount of the FPET RNA is not limiting. If the amount of the FPET RNA islimiting, then the preferred method is to use the oligo dT-T7 primedcDNA, convert it to double-stranded DNA with DNA polymerase I and RNAseH and subsequently amplify it with T7 RNA polymerase and rNTPs. If thesample is to be used for microarray analysis, then the oligo dT-T7primed cDNA is converted to double-stranded DNA as above, andsubsequently amplified with T7 RNA polymerase and biotinylated rNTPs.Again, this protocol allows universal gene expression profiling, usingFPET RNA or, in a more general sense, fragmented RNA of any origin.

An additional protocol of the invention is shown in FIG. 11. In thisprotocol, the RT step is enhanced by using longer reverse primers asshown (10 bases, 20 bases and 30 bases) under otherwise normal RTconditions. The longer primers enable an increase in priming of thefragmented RNAs resulting in more cDNA target for the PCR step. Inaddition, the longer primers may aid in reverse transcription bybridging formalin-modified bases that would otherwise block enzymaticactivity. An additional modification includes performing the initial twocycles of PCR at 50° C. This enables the amplification of more targetcDNA due to the lower annealing temperature. Both of the above stepsresult in stronger gene expression signals.

Further embodiments, provide several additional improvements over theuniversal amplification of fragmented RNA protocol (FRA), as discussedabove. These improved procedures enable global reverse transcription andamplification of smaller quantities (50 ng) of even highly fragmentedFPET RNA samples in an automatable, solid phase bead format. Theimprovements also decrease the number of cleanups between enzymaticsteps involved in the FRA discussed above, making the process a higherthroughput procedure. Furthermore, the improvements permit the archivingof complete fragmented RNA transcriptomes on beads. Although fragmented,the archived RNAs are easily reamplified thus allowing reproduciblemeasurement of mRNA levels of all expressed genes in biopsied orresected tumor tissue and archived paraffin-embedded tissue samples.Finally, the procedure can also easily incorporate an enrichment stepfor mRNA that allows increased sensitivity of gene expression analysis.

A rapid universal FPET RNA amplification procedure should greatlyincrease the number of genes that can be expression profiled and thenumber of studies that can be performed with typically limiting amountsof valuable clinical samples.

The specific improvements and changes to the basic protocol that areincorporated to create the improved bead based protocol are as follows:

-   (a) removal of the cleanup step between deblocking the 3′ termini of    the FPET RNA with PNK and polyadenylating the RNA with EPAP;-   (b) hybridization of the polyadenylated fragmented RNA obtained in    step (a) to a solid phase bead format; the step easily enables an    optional step of enriching for mRNA by removing rRNA sequences    through hybridization prior to step (c);-   (c) conversion of the bead immobilized RNA in step (b) to cDNA and    subsequently to double-stranded DNA;-   (d) amplification of the RNA by subjecting the double-stranded DNA    obtained in step (c) to in vitro transcription with a RNA    polymerase. Performing steps (c) and (d) on beads also decreases    cleanup time between enzymatic steps;-   (e) reduction of the starting FPET RNA sample size from 200 ng to 50    ng;-   (f) ability to archive the the FPET RNA libraries as    bead-immobilized double-stranded DNA and to reamplify the material    to produce additional RNA.

The improvements are illustrated in FIG. 13. The process starts withFPET RNA, generally 50-200 ng, and involves 1) unblocking of the 3′OH onthe terminal nucleotide with PNK, 2) direct EPAP poly A tailing of theFPET RNA 3′ without cleanup from PNK step, 3) hybridization ofpolyadenylated FPET RNA to oligo dT-T7 RNA polymerase promoter sequencesimmobilized to beads followed by 4) cDNA synthesis with RT, 5) partialRNA degradation by RNAse H and second strand DNA synthesis with DNApolymerase I and 6) RNA amplification by in vitro transcription. Anoptional procedure for a second round of IVT is shown in step 7 (brokenarrows). Another optional step, shown in step 2′ involves depletion ofribosomal rRNA fragments (dotted arrow).

The beads used for nucleic acid hybridization can be commerciallyavailable microbeads, such as, for example, Dynal 2.8-μm magneticstreptavidin beads (M-280) or Dynabeads® MyOne™ Streptavidin (DynalBiotech, Oslo, Norway). Streptavidin beads can be easily attached to 5′or 3′ biotinylated nucleic acids. Bead-based immobilized oligo dT hasbeen quite useful in purifying mRNA (Hornes, E. and Korsnes, L. (1990),Genet. Anal. Tech. Appl. 7:145; Jacobsen, K., Breivold, E. and Hornes,E. (1990) Nucleic Acids Res. 18:3669) and for subsequent aRNAamplification (Eberwine, J. (1995), Biotechniques 20:584).

Further details of the invention, including dephosphorylation of the 3′terminus of fragmented RNA, polyadenylation, subsequent reversetranscription using extended primers, and enhanced PCR are illustratedby the following non-limiting Examples.

REFERENCE EXAMPLE 1

In Example 1 below, the following methods were used.

FPET RNA Extraction Procedure

RNA was extracted from 3-10 μm sections (for each patient). Paraffin wasremoved by xylene extraction followed by ethanol wash. RNA was isolatedfrom sectioned tissue blocks using the MasterPure™ Purification kit(Epicentre Technologies, Madison, Wis.) and included a DNase I step.FPET RNA was further purified by filtration through a CHROMA SPIN™DEPC-H20 30 column as described by suppliers (Clontech, Palo Alto).Briefly, 30 μl of 50-300 ng/μl FPET RNA was loaded onto a column(pre-spun at 2500 rpm (664×g) for 5 min. in a 5417 C eppendorfcentrifuge), spun through the column (same conditions as the pre-spin)and stored at −80° C. FIG. 2 shows an example of RNA isolated fromformalin fixed, paraffin embedded (FPE) breast cancer samples that werearchived from 1 to 17 years.

Positive Control Complementary RNA (cRNA) Synthesis

Small RNA fragments complementary to amplicons for the genes HER2,GAPDH, and CYP were generated in two steps: 1) single-stranded DNAfragments complementary to the amplicons for these genes and containinga T7 RNA polymerase site on their 5′ end were synthesized (IDT,Coralville, Iowa) and amplified by PCR. 2) The PCR products werepurified using CHROMA SPIN™ TE-30 columns and cRNA was generated via theAmpliScribe™ T7 Transcription kit (Epicentre Technologies) and purifiedusing CHROMA SPIN™ DEPC-H₂O 30 columns.

Dephosphorylation of the FPET RNA 3 ′ Terminus

The 3′-terminus of the FPET RNA was treated with either T4polynucleotide kinase (PNK) or 0.01M HCl and calf alkaline phosphatase(CIP) to remove 2′-3′ cyclic phosphates, 2′-monophosphates and3′-monophosphates. These various phosphate esters are typically found onthe 3′ terminal ribose moiety of degraded RNAs and need to be removed toensure efficient poly A addition to the 3′ terminus of the FPET RNA.

PNK Treatment

In a 20 μl reaction volume, 100-5000 ng of FPET RNA is incubated at 37°C. for 1 h with 20 units of PNK (NEBiolabs, Beverly, Mass.) in 1×PNKbuffer (70 mM Tris-HCI pH 7.6, 10 mM MgCl₂, 5 mM dithiothreitol) and 40units of RNaseOUT™ (Invitrogen, Carlsbad, Calif.). The reaction isterminated by addition of 20 μl of RNAse free H20 and extraction with 40μl of phenol: CHCl₃: IAA (25:24:1) pH 6.6 (Ambion, Inc., Austin, Tex.).After centrifugation at 14,000×g for 1-2 min., the aqueous phase isremoved, passed over a CHROMA SPIN™ DEPC-H2O 30 column and volumereduced to 12.5 μL using a Savant speed vacuum.

CIP Treatment

In a 20 μl reaction volume, 100-5000 ng of FPET RNA is incubated for 2hrs in 10 mM HCl at 25° C. The FPET RNA is then passed over a CHROMASPIN™ DEPC-H2O 30 column as above and incubated with 10 units of CIP(New England Biolabs, Beverly, Mass.) for 30 min at 37° C. in 1×NEBuffer3 (10 mM NaCl, 5 mM Tris-HCl, pH 7.9, 1 mM MgCl₂, 1 mM dithiothreitol)and 40 units of RNaseOUT™ (Invitrogen, Carlsbad, Calif.). The reactionis terminated by addition of 20 μl of RNAse free H2O and extraction with40 μl of phenol: CHCl₃: IAA (25:24:1) pH 6.6 (Ambion Inc., Austin,Tex.). After centrifugation at 14,000×g for 1-2 min., the aqueous phaseis removed, passed over a CHROMA SPIN™ DEPC-H2O 30 column and volumereduced to 12.5 μL using a Savant speed vacuum.

Polyadenylation of FPET RNA

100-5000 ng of dephosphorylated FPET RNA was incubated at 37° C. (20 μLrxn volume) with 1.0 unit of E. coli poly A polymerase (EPAP) in 1×EPAPbuffer (Ambion Inc., Austin Tex.), 1 mM ATP and 40 units of RNAseOUT™(Invitrogen, Carlsbad, Calif.) for 15 min. The reaction was terminatedby addition of 20 μl of RNAse free H₂O and extraction with 40 μl ofphenol: CHCl₃: IAA (25:24:1) pH 6.6 (Ambion Inc., Austin, Tex.). Aftercentrifugation at 14,000×g for 1-2 min., the aqueous phase was removedand passed over a CHROMA SPIN™ DEPC-H2O 30 column.

FPET RNA Analysis

RNA was quantitated using the RiboGreen fluorescence method (MolecularProbes). RNA size was analyzed by microcapillary electrophoresis usingthe Agilent 2100 Bioanalyzer (Agilent Technologies, Calif.).

TaqMan® Primer/Probe

For each gene, we identified the appropriate mRNA reference sequence(REFSEQ) accession number for the gene and accessed the sequencesthrough the NCBI Entrez Nucleotide database. Primers and probes weredesigned using Primer Express (Applied Biosystems, Foster City, Calif.)and Primer 3 programs [Steve Rozen and Helen J. Skaletsky (2000),Primer3 on the WWW for general users and for biologist programmers. In:Krawetz S, Misener S (eds) Bioinformatics Methods and Protocols: Methodsin Molecular Biology. Humana Press, Totowa, N.J., pp 365-386].Oligonucleotides were supplied by Biosearch Technologies Inc. (Novato,Calif.) and Integrated DNA Technologies (Coralville, Iowa). Ampliconsizes were limited to 85 bases. Fluorogenic probes were dual-labeledwith 5′-FAM and 3′-BHQ1.

Reverse Transcription

Reverse transcription was carried out using a SuperScript™ First-StrandSynthesis Kit for RT-PCR (Invitrogen Corp., Carlsbad, Calif.). Thereactions were carried out with total FPET RNA (3-50 ng/μL) and eitherpooled gene specific primers (100 nM each) or oligo(dT) primers (25ng/μl) or oligo(dT)-T7 primers (0.25-5.0 μM). For the extended primerreverse transcription, the reaction was performed using the OrniscriptReverse Transcriptase for First-strand cDNA synthesis kit (Qiagen,Valencia, Calif.) as described. Total FPET RNA (3-50 ng/μL) and pooledextended gene specific primers were used at concentrations of 3-50 ng/μLand 100 nM (each primer), respectively.

Second Strand DNA Synthesis

1^(st)-strand cDNA synthesis products derived from 100-5000 ng FPET RNAwere incubated at 16° C. (150 μL reaction volume) in 1× second strandbuffer [20 mM Tris-HCl, pH 6.9; 4.6 mM MgCl2; 90 mM KCl; 0.15 mM β-NAD⁺;10 mM (NH₄)₂SO₄], 0.2 mM dNTP mix, 10 units DNA ligase, 40 units DNApolymerase 1, and 2 units RNase H (all reagents Invitrogen, Carlsbad,Calif.) for 2 hours. 9 units T4 DNA polymerase (NEBiolabs) were thenadded; reaction mix was incubated an additional 15 minutes. DNA wasprecipitated with 5M ammonium acetate and 100% ethanol, with 5 μg ofglycogen as a carrier.

In Vitro Transcription (IVT)

The precipitated ds-DNA (from above) was resuspended in 8 μLnuclease-free H₂O and an IVT reaction (20 μL total) was performed usingMEGAscript™ T7 kit (Ambion, Austin Tex.) and allowed to proceed for 4hours at 37° C. Subsequently, reaction volume was increased to 40 μLwith nuclease-free H2O and cRNA was precipitated with 3M sodium acetateand 100% ethanol. Precipitated cRNA was resuspended in 20-40 μLnuclease-free H2O.

TaqMan® Gene Expression Profiling

For ABI 7900® runs, the TaqMan® reactions were performed in duplicate 5μl reactions consisting of 1× Universal PCR Master Mix and cDNA madefrom an equivalent of 1 ng of total RNA. Final primer and probeconcentrations were 0.9 μM (each primer) and 0.2 μM, respectively. PCRcycling was carried out on the ABI Prism® 7900 as follows: 95° C. 10minutes×1 cycle, 95° C. 20 seconds, 60° C. 45 seconds×40 cycles. For7700 runs, the TaqMan® reactions were performed in triplicate 25 μlreactions consisting of 1×PCR buffer A, 4 μM MgCl₂, 0.2 μM dNTPs, 0.025U/μl AmpliTaq Gold™ DNA polymerase (Applied Biosystems, Foster City,Calif.), and cDNA made from an equivalent of 2.5 ng of total RNA. Finalprimer and probe concentrations are as above. PCR cycling was carriedout on an ABI Prism® 7700 as above. For the modified PCR primingexperiments, PCR cycling was carried out on the ABI Prism® 7700 asfollows: 95° C. 10 minutes×1 cycle, 95° C. 20 seconds, 50° C. 2minutes×2 cycles, 95° C. 20 seconds, 60° C. 45 seconds×38 cycles.

EXAMPLE 1

Standard Protocol

RNA was treated with polynucleotide kinase (PNK) or calf intestinalalkaline phosphatase (CIP), enzymes with 2′-3′ cyclic phosphataseactivity and 3′ phosphatase activity, respectively. Capillaryelectrophoretic [Agilent 2100] analysis of the treated FPET RNAsuggested that treatment of the FPET RNA with PNK or CIP removed theblocking phosphates, as judged by a subtle decrease in the mobility ofthe enzyme-treated RNA relative to that of the untreated RNA (FIG. 6A).Decreased electrophoretic mobility was expected because removal of thecharged phosphate group would have decreased the charge/mass ratio ofthe FPET RNA.

If the blocking phosphates from the 3′ end of the FPET RNAs wereeffectively removed, then polyadenylation of the RNA should be possible.Treatment of FPET RNA with PNK followed by EPAP treatment (+PNK/+EPAP)resulted in a significant decrease in electrophoretic mobility of theFPET RNA (FIG. 6B). To confirm that the mobility shift was due topolyadenylation and not simply due to dephosphorylation, PNK treatmentalone (+PNK/−EPAP) and a no treatment (−PNK/−EPAP) controls wereincluded. The only significant decrease in mobility was noticed withboth PNK and EPAP treatment. Thus, the combination of an unblocking,dephosphorylation step (PNK or CIP treatment) followed by apolyadenylation step by EPAP most likely converted the FPET RNA to apolyadenylated form efficiently. This polyadenylated RNA should besuitable for universal cDNA synthesis using oligo dT primers and RT.

To test the effectiveness of polyadenylation on universal cDNAsynthesis, FPET RNA was polyadenylated by EPAP following treatment withor without PNK or CIP, and the cDNA abundance was measured by TaqMan®RT-PCR. PNK (FIG. 7) or CIP (FIG. 8) treatment followed bypolyadenylation and oligo-dT primed RT-PCR resulted in a significantincrease in cDNA yields relative to non-PNK (4-32 fold ) or non-CIP(8-16 fold) treated samples. This indicated that unblocking the 3′ enddramatically increases the efficiency of polyadenylation and oligo dTprimed cDNA synthesis. As expected, polyadenylation had very littleeffect on GSP primed cDNA synthesis. Importantly, the GSP positivecontrols indicated that this universal priming method amplified cDNA25-50% as effectively as the currently most effective priming method,GSP priming.

In a further experiment, cDNA was synthesized from polyadenylated FPETRNA (PNK and EPAP treated) and non-polyadenylated FPET RNA using eitheroligo dT primers or GSP primers, respectively. In this experiment, 96pooled GSP primers were used and expression of 96 genes was analyzed byTaqMan® RT-PCR (using 1 ng FPET RNA/well, 384 wells; ABI Prism® 7900instrument). The data shown in FIG. 9 demonstrate that polyadenylatedFPET RNA was efficiently converted to cDNA (pA-dT) as judged by thesimilarity in Ct values to GSP-primed (pA_GSP1) cDNA. For many genes,the polyadenylated FPET RNA gave a better signal with oligo dT primingthan GSP priming. Table 1 shows a statistical summary of the data fromFIG. 9. The left panel indicates that polyadenylating the FPET RNA priorto RT with oligo dT results in detection of 77% of the genes (Ct <38)whereas nonpolyadenylated RNA yields only 16% detectable genes.Furthermore, there is a significant correlation between the geneexpression profile of cDNA generated by GSP and oligo dT priming ofpolyadenylated RNA (Pearson R=0.77). There was no correlation betweengene expression profiles of cDNA generated by GSP and nonpolyadenylated,oligo dT primed RT (R=0.11).

Another useful improvement to this method was the inclusion of a T7 RNApolymerase site on the oligo dT primer such that FPET RNA could beuniversally amplified following polyadenylation. FIG. 10(A-C)demonstrates the effect of polyadenylation and in vitro transcription(IVT) [T7 RNA polymerase amplification (Van Gelder et al., Proc. Natl.Acad. Sci. USA 87(5):1663-7 (1990)] on the expression of a 46 genes fromthree different RNA sources. FIG. 10A shows expression profiles fromhigh quality intact RNA (Stratagene). IVT increased the average TaqMansignal of all 46 genes (see inset) ˜6 fold when comparing cDNA generatedby GSP primed RT (GSP; non-amplified control) and cDNA generated byoligo dT-T7 primed RT that was subsequently amplified by IVT (No EPAPIVT). Polyadenylation of the RNA prior to cDNA synthesis and IVT had noadditional effect on the overall TaqMan signal (EPAP IVT-1 and IVT-2).FIG. 10B and 10C show gene expression profiles generated from moderatelydegraded FPET RNA (BioPath Placenta) and badly degraded FPET RNA(Clinomics 168), respectively. In these cases, polyadenylation was anecessary step for IVT amplification of the RNA. As shown, the averageTaqMan signals from duplicate experiments (EPAP IVT avg) were ˜2.5 Ctslower (6 fold) than signals generated by IVT from non-polyadenylated RNA(No EPAP IVT). Importantly, the gene profiles are maintained after IVTas indicated by the Pearson correlation coefficient (R=0.91-0.96). Insummary, polyadenylation of degraded FPET RNA is a useful method toglobally synthesize cDNA corresponding to each gene present in thesample. This cDNA can be used to further amplify gene signals accuratelyand reproducibly by IVT.

Another improvement for enhancement of gene expression signals is shownin FIG. 12. In this example, the detection of six genes was enhanced asthe primers were lengthened. Extending the primer lengths to 20-30 basesbeyond the standard reverse primer (GSP) length, increased the geneexpression signals from 10-15 fold. In addition, if the first two cyclesof the subsequent PCR were performed at 50° C. rather than 60° C., thegene expression signals further increased several fold.

REFERENCE EXAMPLE 2

Unless otherwise indicated, in Examples 2-5 below, the followingmaterials and methods were used.

Materials

MyOne Streptavidin-Coated Microspheres: Dynal, 2 mL @ 10 mg/mL.

Biotin-Eberwine Primer: IDT, 100 pmol/uL stock (100 uM).5′-Biotin-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTTTTTTTTTTTTTTVN-3′ (SEQ ID NO: 1)

T4 Polynucleotide Kinase (PNK): New England BioLabs, 2,500 units @ 10U/μL. Comes w/a 10× reaction buffer.

RNase Inhibitor: Applied Biosystems, 20 U/μL.

Nuclease-free H₂O: Ambion.

Poly(A) Tailing Kit: Ambion. Items used in kit—E. Coli poly(A)polymerase (EPAP) enzyme (2 U/μL), 5× reaction buffer, 10 mM ATP.

Superscript RT First-Strand System: Invitrogen. Items used in kit-10×RTbuffer, 0.1M DTT, 10 mM dNTP mix (note: dNTP mix is also used insecond-strand synthesis), 2 U/μL RNase H (used in second strandsynthesis).

Superscript II RT enzyme: Invitrogen, 200 U/μL. Comes with 5×RT buffer,which is used to create 1×RT buffer for pre-RT washing step.

0.1M MgCl₂ : Ambion. Comes as 1M stock and is subsequently diluted 1:105× Second Strand Buffer: Invitrogen.DNA Ligase: Invitrogen, 10 U/μL.

DNA Polymerase I: Invitrogen, 10 U/μL.

T4 DNA Polymerase: Invitrogen, 5 U/μL.

Glycogen: Ambion, 5 mg/mL.

5M ammonium acetate: Ambion.

100% ethanol: SigmaMEGAScript-T7 IVT Kit: Ambion. Items used inkit-10×IVT buffer, 75 mM NTPs, enzyme mix.

3M sodium acetate: Ambion.

Methods

PNK and EPAP Treatment

For 200-300 ng of FPET RNA: FPET RNA was incubated in 1×PNK buffer (70mM Tris-HCl pH 7.6, 10 mM MgCl₂, 5 mM dithiothreitol), 1 U/μl RNaseInhibitor (Applied Biosystems, Foster City, Calif.) and 1 U/μl of PNK(NEBiolabs, Beverly, Mass.) at 37° C. for 30 min in a 20 μl reactionvolume. Following PNK treatment, the FPET RNA was directlypolyadenylated by adding to the reaction mixture to a finalconcentration; 1×EPAP buffer (Ambion, Austin, Tex.), 1 mM ATP, 1.5 U/μlRNase Inhibitor and 0.025 U/μl EPAP (Ambion, Austin, Tex.). The mixturewas incubated at 37° C. for 15 min in a 40 μl reaction volume, then at70° C. for 5 min.

For 50 ng of FPET RNA: PNK and EPAP treatment were identical to above,except for reaction volumes, which were scaled down to 5 μl and 10 μl (¼volume), respectively.

Reverse Transcription of Polyadenylated FPET RNA with T7-Oligo dTPrimer-Magnetic Bead Complex

Preparation of T7-Oligo dT Primer-Magnetic Bead Complexes

Dynabeads® MyOne™ Streptavidin (Dynal Biotech, Oslo, Norway) stock beadcontainer was removed from 4° C. storage and vortexed vigorously tofully resuspend the beads. 40 μL (400 μg) beads were removed to a 0.5 mLmicrocentrifuge tube; spin beads down in a tabletop microcentrifuge (<5sec) to collect liquid in bottom of tube. Avoid over-centrifuging tubescontaining paramagnetic beads, as it will cause them to pellet andaggregate, which can reduce bead performance. Tubes were placed in aMPC-S magnetic rack (Dynal Biotech, Oslo, Norway) with the tube hingesfacing the magnet and allow beads to collect against side of tube (˜2min). Tubes were opened without removing from rack; and the supernatantwas pipetted off. Tubes were removed from rack; beads were washed byresuspending in 100 μL 1×B&W buffer (5 mM Tris-HCl pH 7.3, 0.5 mM EDTA,1M NaCl). Tubes were spinned down briefly to collect liquid, then placedin magnetic tube rack. Beads were allowed to collect against side oftube and supernatant was removed as above; and 1×B&W buffer washrepeated for a total of two washes. Final wash supernatant was removed,then beads were resuspended in 40 uL 1×B&W buffer containing 25 uMEberwine T7 oligo dT primer (5′-BiotinGGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTT TTTTTTTTTTTTTTTTTTTVN-3′).(SEQ ID NO: 1) Incubation was performed at room temperature for 15 minon an Eppendorf thermomixer (850 rpm). Beads were suspended on magnet(˜2 min); supernatant was removed and beads were washed two times w/100μL 1×B&W buffer. Beads were resuspended in 80 μL Bead Storage Buffer(1×PBS, 70% EtOH) and stored at 4° C. until ready for use.

Hybridization of T7-Oligo dT Beads to Polyadenylated FPET RNA

For 200-300 ng of FPET RNA: During PNK/EPAP incubations, the previouslyprepared primer-bead solution was removed from 4° C. storage. The tubewas flicked to resuspend beads and spinned down briefly to collectliquid. 20 μL (100 μg) of beads were aliquoted into a 0.5 mL tube andthe tube placed on magnet for about 2 minutes. The supernatant wasremoved; beads were washed twice with 100 μL 1×B&W Buffer. The finalwash supernatant was removed during the 70° C. EPAP inactivation step.After the 70° C. incubation was completed, the tube containing the EPAPreaction was thoroughly spinned down to collect liquid, then thereaction contents transferred to tube containing primer-bead complex.The tube was flipped to resuspend beads and briefly spinned down;incubated at room temperature on thermomixer (850 rpm) for 5 minutes.The tube was placed on magnetic rack for about 2 minutes; thesupernatant was removed.

For 50 ng of FPET RNA: Same as above except aliquot 5 μL (25 ug) ofbeads.

First-Strand cDNA Synthesis

Reverse transcription was carried out using a SuperScript™ First-StrandSynthesis Kit for RT-PCR (Invitrogen Corp., Carlsbad, Calif.).

For 200-300 ng of FPET RNA: Beads were washed once with 100 μL 1×RTbuffer (20 mM Tris-HCl pH 8.4, 50 mM KCl). Beads were resuspened in 20μL RT reaction mix consisting of 1×RT buffer, 5 mM MgCl₂, 10 mM DTT, 0.5mM dNTPs, 1 U/μL RNase inhibitor, and 10 U/μL SuperScript II RT andincubate at 42° C. on thermomixer (850 rpm) for 50 min.

For 50 ng of FPET RNA: Same as above except beads were resuspended in 5μL 1×RT buffer.

Second-Strand cDNA Synthesis

For 200-300 ng of FPET RNA: First strand cDNA reaction was removed fromthermomixer and 130 μL of the following second strand reaction mixadded: 1.15× second strand buffer, 0.23 mM dNTP mix, 0.077 U/μL DNAligase, 0.31 U/μL DNA polymerase I and 0.015 U/μL RNase H. Flick tube tomix; spin down briefly. Incubate for 2 hrs at 16° C. on thermomixer (850rpm).

For 50 ng of FPET RNA: Same as above except 32.5 μL of the second strandreaction mix is added.

Second-Strand DNA Cleanup/In Vitro Transcription

For both 200/300 ng and 50 ng of FPET RNA: Second-strand reaction tubewas removed from thermomixer; spinned down briefly and place on magneticrack for about 2 minutes. Supernatant was removed and beads washed twicewith 100 μL 1×IVT wash buffer (400 mM Tris pH 7.3, 70 mM MgCl₂, 100 mMNaCl, 20 mM spermidine.

The MEGAScript -T7 IVT kit (Ambion, Austin Tex.) was used for in vitrotranscription. Resuspend beads in 20 μL IVT master mix (prepared asdescribed by Ambion) and flick tube to mix. Spin down briefly andincubate at 37° C. for 4 hrs on thermomixer (1000 rpm).

IVT Cleanup

The tube was removed from thermomixer; spinned down briefly and placedon magnetic rack for about 2 minutes. The supernatant was transferred toa 1.5 mL microcentrifuge tube and the following reagents added in order:Nuclease-free H₂O: 20 μL 5 mg/mL glycogen (Ambion): 1 μL 3 M sodiumacetate (Ambion): 4 μL 100% ethanol (Sigma): 100 μL

The tube was vortexed and incubated @ −20° C. from 1 hr to overnight.The tube was spinned down in a refrigerated Eppendorf centrifuge @14,000 rpm for 20 min (4° C.) to pellet cRNA. The supernatant wasremoved and 500 μL 70% ethanol solution was added to wash pellet. Thetube was spinned down in a refrigerated Eppendorf centrifuge @ 14,000rpm for 3 min (4° C.). With a pipet, as much alcohol was removed fromthe tube as possible, then the tube left (with top open) in a fume hoodto allow remaining alcohol to evaporate (5-10 min). cRNA pellet wasresuspended in 40 μL nuclease-free H₂O.

FPET RNA Analysis

RNA was quantitated using the RiboGreen fluorescence method (MolecularProbes). RNA size was analyzed by microcapillary electrophoresis usingthe Agilent 2100 Bioanalyzer (Agilent Technologies, Calif.).

TagMan® Gene Expression Profiling

TaqMan® reactions were performed in duplicate 5 μl reactions consistingof 1× Universal PCR Master Mix and 1 ng of cRNA. Final primer and probeconcentrations were 0.9 μM (each primer) and 0.2 μM, respectively. PCRcycling was carried out on the ABI Prism® 7900 as follows: 95° C. 10minutes×1 cycle, 95° C. 20 seconds, 60° C. 45 seconds×40 cycles.

EXAMPLE 2

Removal of the Cleanup Step Between Dephosphorylation andPolyadenylation of the FPET RNA 3′ Terminus

The 3′-terminus of the FPET RNA is treated with T4 polynucleotide kinase(PNK) to remove 2′-3′ cyclic phosphates, 2′-monophosphates and3′-monophosphates. These various phosphate esters are typically found onthe 3′ terminal ribose moiety of degraded RNAs and need to be removed toensure efficient poly A addition to the 3′ terminus of the FPET RNA.

As previously described, the standard dephosphorylation step with PNK isterminated by addition of 20 μl of RNAse free H₂O and extraction with 40μl of phenol: CHCl₃: IAA (25:24:1) pH 6.6 (Ambion, Inc., Austin, Tex.).After centrifugation at 14,000×g for 1-2 min., the aqueous phase isremoved, passed over a CHROMA SPIN™ DEPC-H₂O 30 column and volumereduced to 12.5 μL using a Savant speed vacuum. The RNA is now ready forthe standard polyadenylation reaction with EPAP in a 20 μl volume.

To streamline the method, we several modifications to the procedure weretried, as shown in FIG. 2. Following the PNK reaction using 1000 ng ofbreast cancer FPET RNA (Clinomics), six cleanup conditions were testedprior to polyadenylation with EPAP. Cleanup condition 1 is essentiallythe same as the standard protocol discussed earlier, but with the samplevolume reduction step omitted (speed-vac). This results in a largersample volume and thus required an increase in the final EPAP reactionvolume (40 μl). The 40 μl reaction volume was kept constant for all fivecleanup conditions as shown in FIG. 14. Microcapillary electrophoresisanalysis (FIG. 14 left panel) of the polyadenylated RNAs following thefive modified cleanup conditions indicated that on average, 50-140adenylates were added to the RNAs relative to the starting material(SM), which had an average size of 90 nucleotides (nt); For example, forcondition 1; 150−90=60 adenylates. These results indicate that all fiveconditions yielded polyadenylated FPET RNA of acceptable size.Interestingly, the percent of RNA recovered after the EPAP cleanup stepindicated that all five PNK cleanup conditions were better than thestandard method, condition 6 (FIG. 14, lower right). Furthermore, the nocleanup condition (5) gave the highest yield. The percent recovery isrelative to input RNA and in some cases is greater than 100% due to anincrease in mass from polyadenylation. To further assess the quality ofthe polyadenylated RNAs, four samples were carried through the remainderof the standard IVT process and expression profiled by TaqMan® RT-PCR asoutlined in our previous patent application (39740.0003PR). FIG. 15shows a 47 gene panel profile of the amplified RNAs for conditions 2-5.All profiles showed a high concordance (R≧0.91) with unamplified RNA(SM) and standard treated RNA (condition 6). The no cleanup condition 5gave the lowest average Ct (36.0) and the highest IVT yield (95.8 μg)and thus was adopted as the standard procedure for EPAP treatment (step2, FIG. 13).

EXAMPLE 3

Hybridization of Polyadenylated FPET RNA to T7 Promoter-Oligo dT Beads,Double-Strand cDNA Synthesis and IVT.

Another improvement to the standard method of the present inventionincludes the hybridization of the polyadenylated FPET RNA to T7promoter-oligo dT primers that are conjugated to magnetic polystyrenebeads. This enables all subsequent enzymatic steps to be easilyperformed on a solid support with minimal cleanup steps. For instance,it eliminates the need for phenol-CHCl₃ extractions and spin columnchromatography between reactions. The use of beads also lends itself toautomated process that could greatly increase the throughput. Inaddition, archived, bead conjugated-cDNA libraries can be easilyre-amplified to yield additional cRNA. FIG. 16 shows the 47 geneexpression profile for placental cRNAs generated by the standardsolution, non-bead based IVT method (free IVT) and cRNA generated by thesolid phase, bead based IVT method (solid-phase IVT) outlined in FIG.13. For comparison, non-amplified placental FPET RNA is also shown (SM).Again, both IVT processes yielded cRNA that displayed a high concordancewith untreated RNA (R≧0.94). Although the traditional non-bead IVTmethod yielded 1.74 times more cRNA than the bead based IVT method, theaverage Ct was 0.74 higher. When the amounts of RNA are adjusted toyield the same average Ct as starting material RNA, the relative yields(Ct adjusted) are approximately equal (21.66 vs. 20.79). Thus, the beadbased IVT method is nearly as efficient as the non-bead based method interms of amplification and fidelity. Although both IVT methods do notachieve the same level of RT-PCR sensitivity (avg. Ct/mass RNA) asstarting material when using equivalent amounts of RNA (1 ng/well), theystill are achieving a 100-fold amplification of RNA after adjusting forthe loss of sensitivity.

EXAMPLE 4

A Comparison of RNA Amplification Using 50 ng and 200 ng FPET RNA

Archived FPET samples with clinical histories are extremely valuable forretrospective clinical studies. As such, it is often difficult to obtainmore than 1 or 2 five-micron FPET sections per patient from clinicalcollaborators for studies. Thus, there is a need to optimize IVTamplification of FPET RNA starting from less than a microgram of RNA andpreferably less than 100 ng RNA. FIG. 17 shows the 47 gene expressionprofile from 200 ng (condition 1) and 50 ng (conditions 2 and 3) of FPETRNA amplified by the bead-based protocol. For the first 50 ng RNAamplification (condition 2), the ratio of RNA to bead mass and thereaction volumes of each step were identical to the standard 200 ng RNAamplification. For the second 50 ng RNA amplification (condition 3), theratio of RNA to bead mass was identical, but the reaction volumes werescaled down proportionally to ¼ of the volume. Both 50 ng RNAamplications yielded approximately the same amount of cRNA, although10-fold less than the 200 ng RNA amplification. If we expect a 4-foldlower yield from the 50 ng reactions, since we started with ¼ the amountof FPET RNA, then we still have an unaccounted 2.5-fold loss inefficiency when scaling down our amplication from 200 ng to 50 ng ofFPET RNA. However, both expression profiles derived from the 50 ngamplication still show a strong correlation with the 200 ng RNAamplication (R≧0.97). Also, the average Ct for the 50 ng volume-scaledamplification (condition 3) was equivalent to the 200 ng RNAamplification. Thus, the scaled down (¼ vol) version was adopted as the50 ng FPET RNA amplification procedure.

EXAMPLE 5

A Comparison of a Secondary Amplification Using 50 ng and 200 Nanogramof FPET RNA

As previously mentioned, an additional benefit of archiving cDNA FPETlibraries on beads is that they can be easily reamplified. As an exampleof a secondary IVT, archived beads containing conjugated cDNA from theabove experiment, were washed once, resuspended in IVT buffer andamplified according to the original IVT protocol. FIG. 17 b shows theresults of this experiment. Again, both 50 ng RNA amplications yieldedapproximately the same amount of cRNA, although 10-fold less than the200 ng RNA amplification. In addition, all three secondaryamplifications yielded about ⅓ as much RNA as their correspondingprimary amplifications. A high level of fidelity was maintained betweenthe three secondary amplifications (R≧0.95). The primary and secondaryamplifications for each individual condition also maintained a highlevel of concordance (R≧0.97). The expression profiles for the condition3 (FRA-¼ vol: 50 ng/25 ug) primary and secondary IVTs are shown in FIG.17 c.

All references cited throughout the present disclosure are herebyexpressly incorporated by reference.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. Indeed, the present invention is inno way limited to the methods and materials described. For purposes ofthe present invention, the following terms are defined below.

1. A method for preparing fragmented RNA comprising a multiplicity ofRNA species for gene expression analysis comprising the steps of: (a)polyadenylating the fragmented RNA, and (b) converting thepolyadenylated fragmented RNA obtained in step (a) to cDNA.
 2. Themethod of claim 1 wherein the size of the RNA species within thefragmented RNA is between about 20 bases and about 2000 bases.
 3. Themethod of claim 1 wherein the average size of the RNA species within thefragmented RNA is about 50 and about 300 bases.
 4. The method of claim 1wherein polyadenylation is performed with E. coli polyA polymerase. 5.The method of claim 1 additionally comprising the step of deblocking the3′ termini of the fragmented RNA species prior to step (a) with adeblocking agent.
 6. The method of claim 5 wherein said deblocking agentis a phosphatase enzyme.
 7. The method of claim 6 wherein saidphosphatase is selected from the group consisting of calf alkalinephosphatase (CIP), bacterial alkaline phosphatase, shrimp alkalinephosphatase, and variants thereof.
 8. The method of claim 5 wherein saiddeblocking agent is a polynucleotide kinase (PNK), or a variant thereof.9. The method of claim 8 wherein said polynucleotide kinase is T4polynucleotide kinase (T4 PNK), or a variant thereof.
 10. The method ofclaim 5 wherein said deblocking agent is removed prior to performing thepolyadenylation step (a).
 11. The method of claim 5 whereinpolyadenylation step (a) is performed without prior removal of thedeblocking agent.
 12. The method of claim 11 wherein said deblockingagent is a polynucleotide kinase (PNK).
 13. The method of claim 1wherein the polyadenylated fragmented RNA obtained in step (a) isconverted to cDNA by treatment with a reverse transcriptase and oligo-dTprimers.
 14. The method of claim 13 wherein said reverse transcriptaseis selected from the group consisting of avian myeloblastosis virusreverse transcriptase (AMV-RT), Moloney murine leukemia virus reversetranscriptase (MMLV-RT), and recombinant heterodimeric reversetranscriptases expressed in E. coli.
 15. The method of claim 14 whereinsaid oligo-dT primers contain an RNA polymerase promoter sequence. 16.The method of claim 15 wherein said promoter is a T7 RNA polymerasepromoter.
 17. The method of claim 13 wherein said polyadenylatedfragmented RNA obtained in step (a) is immobilized prior to conversionto cDNA.
 18. The method of claim 17 wherein said immobilization isperformed on beads.
 19. The method of claim 18 wherein the immobilizedpolyadenylated fragmented RNA is enriched prior to conversion to cDNA.20. The method of claim 19 wherein the enrichment comprises removal ofrRNA sequences by hybridization to bead immobilized complementary rRNAoligonucleotides.
 21. The method of claim 13 wherein the RNA is mRNAobtained from a fixed, paraffin-embedded tissue sample.
 22. The methodof claim 21 wherein said tissue sample is from a tumor.
 23. The methodof claim 22 wherein said tumor is cancer.
 24. The method of claim 23wherein said cancer is selected from the group consisting of breastcancer, colon cancer, lung cancer, prostate cancer, hepatocellularcancer, gastric cancer, pancreatic cancer, cervical cancer, ovariancancer, liver cancer, bladder cancer, cancer of the urinary tract,thyroid cancer, renal cancer, carcinoma, melanoma, and brain cancer. 25.The method of claim 15 further comprising the step of (c) PCRamplification using one or more cDNA species present in the cDNAobtained in step (b) as a template.
 26. The method of claim 25 whereinPCR amplification comprises 40 cycles, and the first five cycles areperformed at a lower annealing/extension temperature.
 27. The method ofclaim 26 wherein PCR amplification comprises 40 cycles, and the firsttwo cycles are performed at a lower annealing/extension temperature. 28.The method of claim 25 wherein PCR amplification comprises 40 cycles,and the first two to five cycles are performed at a temperature of about40° C. to 58° C.
 29. The method of claim 28 wherein said early cyclesare performed at a temperature of about 50° C.
 30. The method of claim15 further comprising the steps of: (d) converting the cDNA obtained instep (b) to double-stranded DNA; and (e) amplifying the RNA bysubjecting the double-stranded DNA obtained in step (d) to in vitrotranscription with an RNA polymerase to obtain amplified complementaryRNA (cRNA).
 31. The method of claim 30 wherein in step (d) the cDNA isconverted to double-stranded DNA using RNaseH and DNA polymerase I. 32.The method of claim 30 wherein the amplified cRNA from step (e) is useddirectly as a template in a gene expression profiling assay by RT-PCR.33. The method of claim 30 wherein step (e) includes labeling of theamplified cRNA with a detectable label.
 34. The method of claim 33wherein the detectable label is biotin or a fluorescent label.
 35. Themethod of claim 30 comprising subjecting the amplified cRNA obtained instep (e) to hybridization to a microarray.
 36. The method of claim 1wherein the polyadenylated fragmented RNA obtained in step (a) isconverted to cDNA by treatment with a reverse transcriptase and extendedreverse primers, and the cDNA obtained is amplified by PCR usingnon-extended forward and a reverse PCR primers and probes, designedbased on target amplicons.
 37. The method of claim 36 wherein theextended reverse primers extend 10 bases further into the amplicon thanthe gene specific reverse PCR primer.
 38. The method of claim 36 whereinthe extended reverse primers extend 20 bases further into the ampliconthan the gene specific reverse PCR primer.
 39. The method of claim 36wherein the extended reverse primers extend 30 bases further into theamplicon than the gene specific reverse PCR primer.
 40. The method ofclaim 36 wherein the extended reverse primers extend into the ampliconwithin 1 base of the forward PCR primer.
 41. A method for enhanced cDNAsynthesis comprising converting RNA to cDNA by treatment with a reversetranscriptase and extended primers, and amplifying the cDNA obtained byPCR using a forward and a reverse PCR primer and a probe, designed basedon a target amplicon.
 42. The method of claim 41 wherein said RNA isfragmented.
 43. The method of claim 42 wherein at least part of saidfragmented RNA is non-polyadenylated.
 44. The method of claim 43 whereinthe extended reverse primers extend 10 bases further into the ampliconthan the reverse PCR primer (GSP).
 45. The method of claim 43 whereinthe extended reverse primers extend 20 bases further into the ampliconthan the reverse PCR primer (GSP).
 46. The method of claim 43 whereinthe extended reverse primers extend 30 bases further into the ampliconthan the reverse PCR primer (GSP).
 47. The method of claim 43 whereinthe extended reverse primers extend to within 1 base of the forward PCRprimer.
 48. A method for preparing RNA comprising a multiplicity of RNAspecies for gene expression analysis comprising the steps of: (a)polyadenylating said RNA; and (b) converting the polyadenylated RNA tocDNA by reverse transcriptase and oligo dT or oligo dT-T7 primers. 49.The method of claim 48 wherein said RNA comprises RNA species withblocked 3′-termini.
 50. The method of claim 49 comprising deblockingsaid blocked RNA species prior to step (a).
 51. The method of claim 48further comprising the step of using the oligo dT primed cDNA directlyfor gene expression analysis.
 52. The method of claim 51 wherein saidgene expression analysis is performed by TaqMan®.
 53. The method ofclaim 48 further comprising the step of converting the oligo dT-T7primed cDNA to double-stranded DNA with DNA polymerase I and RNAse H,and amplifying the double-stranded DNA with T7 RNA polymerase and rNTPs.54. The method of claim 48 further comprising the step of converting theoligo dT-T7 primed cDNA to double-stranded DNA, and amplifying thedouble-stranded DNA with T7 RNA polymerase and biotinylated rNTPs.
 55. Amethod for preparing RNA comprising a multiplicity of RNA species forgene expression analysis comprising the steps of: (a) polyadenylatingsaid RNA; and (b) converting the polyadenylated RNA to cDNA by reversetranscriptase and oligo dT-T7 primers containing a T7 RNA polymerasepromoter, and (c) subjecting the double-stranded DNA obtained in step(b) to in vitro transcription with a T7 RNA polymerase to obtainamplified complementary RNA (cRNA).